Characterization and cardiac differentiation of chicken spermatogonial stem cells

Abstract

Spermatogonial stem cells (SSCs), a unique population of germline stem cells in adult testis, have the capability to self-renew and produce daughter cells destined to differentiate into spermatozoa throughout the life of the bird. Chicken SSCs were successfully isolated from testicular cells and subsequent analysis was performed to identify pluripotent cells by investigation with cytochemical reagents including Periodic acid-Schiff (PAS), alkaline phosphatase (AP), and antibodies to germline cell specific (DAZL or VASA) and stage-specific embryonic antigens (Oct4, SSEA1, SSEA3, SSEA4, TRA-1-60, and TRA-1-81). Results confirmed these as germline cells with the expression of DAZL (Deleted in Azoospermia-Like) and VASA genes in isolated cells. Immunochemistry results showed that multipotent germline stem cells (mGSCs) expressed these gene markers related to embryonic stem cells (ESCs) and could spontaneously differentiate into three embryonic germ (EG) layers in vitro. The mGSC-derived cardiomyocytes expressed cardiac-specific markers such as sarcomeric alpha actinin, alpha-cardiac actinin; conexin-43, the major protein of gap junctions which are thought to have an important role in the synchronized contraction of the heart and in embryonic development; and cardiac troponin T, the tropomyosin binding subunit of the troponin complex which regulates muscle contraction. Furthermore, reverse transcription polymerase chain reaction (RT-PCR) results indicated that the genes related to cardiac transcription factors were expressed following differentiation. Results of the present study strongly contribute to the information related to the ability of chicken mGSCs to differentiate into cells such as contraction cardiomyocytes similar to ESCs and may provide a new source of cardiomyocytes for basic research and potential therapeutic application in various cardiac degenerative diseases of birds and other animals.